Cancer-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin and immunoglobulins. Moreover, biomarkers in the blood may be subjected to degradation during transportation and storage. Such degradation is a significant source of bias for cancer biomarker measurement and discovery. We have created N-isopropylacrylamide porous sieving core shell "smart" nanoparticles containing an internal affinity bait to perform three independent functions within minutes, in one step, in solution (serum or plasma): a) molecular size sieving with complete separation from high abundance residence proteins such as albumin and immunoglobulin, b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis. In our preliminary studies we have manufactured large quantities of uniform porous nanoparticles containing specific bait chemistries that bind highly labile low abundance target cancer biomarker proteins (e.g., platelet derived growth factor). We have demonstrated that the particles can capture and concentrate target proteins from serum such that the target molecule is completely removed from the bulk solution within 5 minutes, with completed exclusion of albumin and immunoglobulins. The captured proteins, even if denatured, are protected from degradation by high concentrations of proteases (trypsin or chymotrypsin) even at 37 oC overnight. The deliverable goal of this proposed feasibility R21 study is to combine this "smart" nanoparticle technology with standard blood collection tubes, with or without additives/anticoagulants, to rapidly encapsulate cancer biomarkers known to be very rare and labile. The envisioned technology is a panel of dry lyophilized sub-micron sized harvesting particles that carry specific bait for known biomarkers. Following introduction of the blood or body fluid, the respective particle populations will remove all of their target molecules, in one step, in solution, from the entire volume of the sample and concentrate the sequestered analytes inside the particles. Analytes will then be eluted from the particles to yield a much higher concentration and purification compared to the starting sample. Depending on the starting volume of the blood, this technology can concentrate a biomarker many hundred fold, and prevent degradation, within minutes. PUBLIC HEALTH RELEVANCE: Biomarkers for early stage cancer are urgently needed so that treatment can be initiated prior to metastasis. Unfortunately, cancer biomarkers exist in very low concentrations and are highly labile. This project will deliver a novel nanotechnology that can rapidly harvest, concentrate, and protect from degradation, labile biomarkers in whole blood or serum, in one step, in the blood collection tube.